Abstract The replication fork helicase unwinds genomic DNA at a replication fork. The assembly and activation of the eukaryotic replication fork helicase is highly regulated. Cdc45, Mcm2-7, and GINS (CMG) form a large assembly that is the active helicase, and the Mcm2-7 is the heterohexameric ATPase that forms the motor of the CMG. The assembly and activation of the CMG is governed by two essential S-phase kinases (S-CDK and DDK), and four essential initiation factors (Sld2, Sld3, Dpb11, and Mcm10) in budding yeast. S-CDK and DDK are currently investigated as targets for the development of cancer chemotherapeutic agents, and Mcm2-7 proteins serve as tumor markers. The Mcm2-7 loads as a double hexamer in late M and G1 phases, and in S phase the Mcm2-7 rings dissociates to single hexamers (Figure 1). Critical unanswered question in the initiation of DNA replication are: (1) How is the Mcm2-7 ring opened during S phase to allow for the extrusion of ssDNA (i.e. origin melting)? (2) How is origin DNA melted? and (3) How is melted origin DNA transferred to RPA, the eukaryotic single-stranded binding protein? Our central hypotheses are that DDK and S-CDK activity function with the essential initiation factors, Sld2, Sld3, Dpb11 and Mcm10, to open the Mcm2-7 ring, melt origin DNA, stabilize melted origin single-stranded DNA, and transfer melted origin DNA to RPA. We have also reconstituted a DNA replication initiation assay using purified budding yeast proteins, and we have generated or acquired conditional degron strains for each of the replication proteins. Thus, we will use a combination of in vitro reconstitution assays and in vivo experiments to test our hypotheses. We will first determine the Mcm2-7 subunit interface required for origin melting during S phase. We will also determine whether Mcm2-7 ring opening is required for subsequent CMG assembly or Mcm2-7 double-hexamer dissociation, or whether CMG assembly and double-hexamer dissociation occur prior to Mcm2-7 ring opening. Thus, we will establish the sequence of key events required for DNA replication initiation. We will also test the hypothesis that S-CDK and DDK phosphorylate Mcm2-7 proteins to promote origin melting during S phase. Sld2, Sld3, Dpb11, or Mcm10 each has biochemical activity for binding origin ssDNA. We will determine how Sld2, Sld3, Dpb11, and Mcm10 function with one another to stabilize single-stranded DNA as it is produced during the process of origin melting. Our hypothesis is that Mcm10 or Sld2-Sld3-Dpb11 function in a S-CDK-dependent manner coordination to stabilize melted origin DNA, preventing reannealing to double-stranded DNA. Finally, we will determine how the melted origin DNA is ultimately transferred to RPA. We have preliminary data suggesting that Dpb11 interaction with RPA is required for DNA replication, and we propose that Dpb11 hands-off melted single-stranded DNA to RPA at a replication origin. Taken together, these three aims will provide a comprehensive view of how cell cycle kinases function with replication initiator proteins to mediate replication fork helicase activation and DNA replication initiation in eukaryotes.